Myostatin is a major player in the regulation of mammalian muscle growth and development, maintaining the balance between proliferation and differentiation prenatally and the quiescence of satellite cells in adults. An absence or overexpression of myostatin results in double-muscling and cachexia respectively, placing myostatin as a promising target in the treatment of muscle wastage diseases. As a transforming growth factor-β superfamily member, myostatin is produced as a precursor protein, consisting of a propeptide region N-terminal to the growth factor domain. Cleavage of the precursor between the domains forms the myostatin latent complex, an inhibitory structure which is exported from the cell where a second cleavage event releases the active myostatin growth factor. The precursor protein, propeptide, and latent complex play important roles in the regulation of myostatin. However, their structure and function are poorly understood, and a possible role for the myostatin precursor protein in the muscle wastage disease sporadic inclusion body myositis, suggests that pre-growth factor forms of myostatin may be additional important therapeutic targets. This thesis presents an investigation into the structure and function of the myostatin precursor protein, the latent complex, and the propeptide region within these, with comparisons to a mutant form of myostatin responsible for the naturally-occurring double-muscled phenotype of the Piedmontese cattle breed. Results suggest that the diverse functions of the propeptide region are facilitated by regions of intrinsic disorder within a primarily structured domain, and that conformational alterations accompany the precursor to latent complex transition, resulting in a tighter inhibitory structure. Comparative analyses between the wild-type and mutant proteins suggest that the Piedmontese phenotype is due to a reduced capacity for covalent dimerisation and significant structural alterations within the type I receptor-binding domain. Investigation into misfolded myostatin precursor protein found that the precursor is able to form cytotoxic amyloid aggregates and mature fibrils under partially denaturing conditions, suggesting a possible mechanism for the role of the myostatin precursor in sporadic inclusion body myositis. Together, these novel results contribute important information towards an understanding of myostatin structure, function and regulation in both normal and disease scenarios.

Myostatin is a major player in the regulation of mammalian muscle growth and development, maintaining the balance between proliferation and differentiation prenatally and the quiescence of satellite cells in adults. An absence or overexpression of myostatin results in double-muscling and cachexia respectively, placing myostatin as a promising target in the treatment of muscle wastage diseases. As a transforming growth factor-β superfamily member, myostatin is produced as a precursor protein, consisting of a propeptide region N-terminal to the growth factor domain. Cleavage of the precursor between the domains forms the myostatin latent complex, an inhibitory structure which is exported from the cell where a second cleavage event releases the active myostatin growth factor. The precursor protein, propeptide, and latent complex play important roles in the regulation of myostatin. However, their structure and function are poorly understood, and a possible role for the myostatin precursor protein in the muscle wastage disease sporadic inclusion body myositis, suggests that pre-growth factor forms of myostatin may be additional important therapeutic targets. This thesis presents an investigation into the structure and function of the myostatin precursor protein, the latent complex, and the propeptide region within these, with comparisons to a mutant form of myostatin responsible for the naturally-occurring double-muscled phenotype of the Piedmontese cattle breed. Results suggest that the diverse functions of the propeptide region are facilitated by regions of intrinsic disorder within a primarily structured domain, and that conformational alterations accompany the precursor to latent complex transition, resulting in a tighter inhibitory structure. Comparative analyses between the wild-type and mutant proteins suggest that the Piedmontese phenotype is due to a reduced capacity for covalent dimerisation and significant structural alterations within the type I receptor-binding domain. Investigation into misfolded myostatin precursor protein found that the precursor is able to form cytotoxic amyloid aggregates and mature fibrils under partially denaturing conditions, suggesting a possible mechanism for the role of the myostatin precursor in sporadic inclusion body myositis. Together, these novel results contribute important information towards an understanding of myostatin structure, function and regulation in both normal and disease scenarios.

Myostatin is a major player in the regulation of mammalian muscle growth and development, maintaining the balance between proliferation and differentiation prenatally and the quiescence of satellite cells in adults. An absence or overexpression of myostatin results in double-muscling and cachexia respectively, placing myostatin as a promising target in the treatment of muscle wastage diseases. As a transforming growth factor-β superfamily member, myostatin is produced as a precursor protein, consisting of a propeptide region N-terminal to the growth factor domain. Cleavage of the precursor between the domains forms the myostatin latent complex, an inhibitory structure which is exported from the cell where a second cleavage event releases the active myostatin growth factor. The precursor protein, propeptide, and latent complex play important roles in the regulation of myostatin. However, their structure and function are poorly understood, and a possible role for the myostatin precursor protein in the muscle wastage disease sporadic inclusion body myositis, suggests that pre-growth factor forms of myostatin may be additional important therapeutic targets. This thesis presents an investigation into the structure and function of the myostatin precursor protein, the latent complex, and the propeptide region within these, with comparisons to a mutant form of myostatin responsible for the naturally-occurring double-muscled phenotype of the Piedmontese cattle breed. Results suggest that the diverse functions of the propeptide region are facilitated by regions of intrinsic disorder within a primarily structured domain, and that conformational alterations accompany the precursor to latent complex transition, resulting in a tighter inhibitory structure. Comparative analyses between the wild-type and mutant proteins suggest that the Piedmontese phenotype is due to a reduced capacity for covalent dimerisation and significant structural alterations within the type I receptor-binding domain. Investigation into misfolded myostatin precursor protein found that the precursor is able to form cytotoxic amyloid aggregates and mature fibrils under partially denaturing conditions, suggesting a possible mechanism for the role of the myostatin precursor in sporadic inclusion body myositis. Together, these novel results contribute important information towards an understanding of myostatin structure, function and regulation in both normal and disease scenarios.

The aim of this thesis was to analyze the polymorphism in position -371bp (related to ATG start codone) of MSTN gene, SNP in position 1984bp of MYF5 gene and influence of these polymorphisms on tenderness, water holding capacity, pH and color of meat. Samples (241) of bull meat of Czech pied cattle were used for analysis. PCR-RFLP method was applied to genotype MSTN and MYF5 genes. For polymorphisms detection was used restriction endonucleases DraI (for MSTN gene) and TaqI (for MYF5 gene). From established genotypes were computed their frequencies and alleles frequencies. The frequencies of genotypes in MSTN gene were AA=0,729, AB=0,258, BB=0,013 and alleles frequencies were A=0,858, B=0,142. The frequencies of genotypes in MYF5 gene were AA=0,181, AB=0,542, BB=0,278 and alleles frequencies were A=0,452 a B=0,548. Using statistical analysis, the influence of genotypes of MYF5 gene on the water holding capacity, pH and the color of meat was determined.

La masse musculaire est largement régulée par des voies de signalisation contrôlant l'équilibre entre la synthèse et la dégradation des protéines myofibrillaires. Ainsi, la myostatine, membre de la superfamille des TGFβ, cible un certain nombre de réseaux de signalisation impliqués dans la régulation de la masse musculaire, notamment la voie de signalisation Akt/mTOR. La myostatine est un des inhibiteurs majeurs de la myogenèse en exerçant un contrôle négatif sur la prolifération et différenciation des myoblastes. A l’heure actuelle, la myostatine est au centre de nombreuses stratégies thérapeutiques notamment dans le cadre de thérapies visant à améliorer la fonction musculaire dans les cas d’atrophie ou myopathies. Les protéines GASP-1 et GASP-2, deux protéines sécrétées contenant plusieurs domaines associés à des inhibiteurs de protéases, ont été décrites comme antagonistes de la myostatine. L’Unité de Génétique Moléculaire Animale a mis en place des stratégies in vitro et in vivo afin de déterminer les fonctions des protéines GASP, notamment dans le contexte myogénique. Dans un premier temps, l'équipe a généré une lignée de souris transgéniques TgGasp-1 sur-exprimant le gène Gasp-1. Cette lignée présente une augmentation globale du poids des muscles squelettiques et une hypertrophie, conséquences d'une inhibition de la myostatine. Cependant, contrairement aux souris knock-out pour la myostatine (Mstn-/-), cette lignée ne présente nid’hyperplasie, ni de changement dans la proportion des différents types de fibres musculaires, ni de variation de métabolisme.Afin de mieux comprendre le rôle des protéines GASP dans le développement musculo-squelettique,deux approches complémentaires ont été développées au cours de cette thèse.Une première approche in vivo, avec l'étude d'une lignée murine TgGasp-2 sur-exprimant le gène Gasp-2, a permis de mettre en évidence un phénotype musculaire semblable aux souris sur-exprimant Gasp-1. En effet, l'analyse phénotypique de ce modèle murin montre à 12 semaines, une augmentation globale du poids des souris et de certains muscles squelettiques due à une hypertrophie des fibres musculaires. Comme pour les souris sur-exprimant Gasp-1, à la différence des souris Mstn-/-, le nombre total de fibres des souris TgGasp-2 et leur métabolisme ne présentent pas de variation parrapport aux souris sauvages. Une seconde approche in silico, suite à une étude transcriptomique et protéomique à partir de modèles murins sur-exprimant ou non GASP-1, a permis d'identifier différents processus biologiques et voies de régulation contrôlées par GASP-1. / Muscle mass is largely regulated by signaling pathways controlling the balance between synthesis and degradation of myofibrillar proteins. Thus, myostatin, a member of the TGFβ superfamily, targets a number of signaling networks involved in the regulation of muscle mass, in particular the Akt / mTOR signaling pathway. Myostatin is one of the major inhibitors of myogenesis by exerting a negativecontrol on the proliferation and differentiation of myoblasts. Today, myostatin is involved in many therapeutic strategies which aim to improve muscle function in cases of atrophy or myopathies.GASP-1 and GASP-2 are two secreted proteins containing several domains associated with protease inhibitors, and described as myostatin antagonists. The Animal Molecular Genetics laboratory has developed in vitro and in vivo strategies to determine the functions of GASPs proteins in a myogenic context. First, we generated a transgenic mouse line TgGasp-1 over-expressing the Gasp-1 gene. This line shows an overall increase in skeletal muscle weight and hypertrophy, a consequence of myostatin inhibition. However, unlike myostatin knockout mice (Mstn -/-), this line shows neither hyperplasia, nor change in the proportion of different types of muscle fibers. Moreover, the global metabolism is not affected. In order to better understand the role of GASPs proteins in musculoskeletal development, two complementary approaches were developed during this thesis :(i) the study of a murine TgGasp-2 line over-expressing Gasp-2 reveals a muscular phenotype similar to the TgGasp-1 mice. At 12 weeks, we observed an overall increase in body and some skeletal muscles weight due to a hypertrophy of the myofibers. As the TgGasp-1 mice, and unlike the Mstn -/- mice, the number of fibers and the metabolism of TgGasp-2 mice did not vary compared to the wildtype mice (ii) In silico analyses allow us to identify different biological processes and regulated pathways controlled by GASP-1.

HMb, metabólito da leucina, utilizado para aumentar a síntese protéica. Investigamos o efeito do HMb sobre o eixo somatotrófico, bem como o mRNA de IGF-I e miostatina muscular. Ratos tratados com HMb (320 mg/Kg de peso corporal /mL de salina-0,9%), ou salina (controle), gavagem, 4 semanas, decapitados, sangue para avaliação sérica: insulina (RIE), glicose (colorimetria) e IGF-I (RIE). Extração de RNA total, para avaliação do mRNA de IGF-I e miostatina (Fígado, músculo extensor digital longo, Sóleo), avaliação da expressão do mRNA do GH, por Northern Blot, e expressão do GH ,Western blotting (hipófise). Dados analisados pelo teste-T de Student (P<0,05). Tratamento aumentou o conteúdo de mRNA de GH (> 60%), da proteína GH (>20%), do mRNA do IGF-I (~24%), da concentração sérica de IGF-I (p<0,05), indicando uma ativação do eixo somatotrófico pelo HMb, sem alterações no mRNA de miostatina e IGF-I muscular, ainda um aumento da insulina (~2x), sem alterações na glicose sérica, resultado do efeito hiperglicemiante do GH, ou um efeito direto do HMb na secreção de insulina. / HMb, metabolite of leucine, used to increase protein synthesis. Evaluate the effect HMb on the somatotrophic axis activity, as well as muscle mRNA IGF-I and myostatin. Rats treated with HMb (320 mg / kg body weight / mL of saline-0, 9%) or saline (control), gavage, 4 weeks, decapitated, blood for evaluation of serum: insulin (RIA), glucose (colorimetric) and IGF-I (RIA). Extraction of RNA total, for evaluation the mRNA IGF-I and myostatin (liver, muscle extensor digitalis longus (EDL) and soleus), evaluation of the GH mRNA expression of by Northern blot, and GH content, western blotting (pituitaries). Data analyzed by Student t-test (P <0.05). HMb treatment increased the content of GH mRNA (> 60%), GH (> 20%), IGF-I mRNA (~ 24%), IGF-I (p <0.05), indicates that the somatotrophic axis activity is increased by the HMb, without changes in mRNA of myostatin and muscle IGF-I, insulin also increased (~ 2x), without changes in serum glucose, hyperglycemiant result of the effect of GH or a direct effect of HMb in the secretion of insulin.

Orientador: Lucia Elvira Alvares / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T02:44:23Z (GMT). No. of bitstreams: 1 Grade_CarlaVermeulenCarvalho_M.pdf: 54304678 bytes, checksum: 869f67d6a836e256bbd7ee9e15980659 (MD5) Previous issue date: 2009 / Resumo: A proteina Miostatina (tambem conhecida como GDF8) e um membro da superfamilia de crescimento e diferenciacao ß (TGF- ß) e e expressa quase que exclusivamente em musculatura esqueletica, tanto no embriao em desenvolvimento quanto no individuo adulto, onde circula livre pela corrente sanguinea. A Miostatina foi inicialmente identificada em 1997 por MCPHERRON et al. e, desde entao, muitos estudos tem demonstrado seu papel essencial na regulacao do desenvolvimento de musculatura esqueletica de aves e mamiferos. O nocaute genico da Miostatina causa hiperplasia e hipertrofia das fibras musculares, resultando em musculos individuais ate duas vezes maiores do que em animais selvagens. Isso demonstra que a Miostatina e um regulador negativo da deposicao de musculatura esqueletica. A estrutura e a funcao desta proteina sao conservadas em diversas especies, incluindo humanos, onde os niveis de Miostatina circulante no sangue se encontram aumentados durante condicoes de distrofia e na caquexia que acompanha alguns tipos de cancer e a AIDS. Um melhor entendimento dos mecanismos que regem a expressao da Miostatina e essencial para o desenvolvimento de estrategias que possam regular sua atividade durante tais condicoes. No presente trabalho, nos identificamos, com o uso de ferramentas de Bioinformatica, elementos cisregulatorios putativos (promotor e enhancers) que possivelmente regulam a transcricao do gene da Miostatina. Inicialmente foi realizada uma comparacao dos loci do GDF8, incluindo as regioes intergenicas adjacentes, provenientes dos genomas de Humano, Camundongo e Galinha. Essa analise revelou a presenca de diferentes regioes evolutivamente conservadas (RECs) adjacentes a sequencia codificadora desta proteina, sete downstream e uma upstream ao gene. Por terem sido mantidas relativamente conservadas ao longo da evolucao, essas regioes supostamente possuem um papel funcional, possivelmente como elementos cis-regulatorios do gene da Miostatina. Em seguida, com o intuito de entender as funcoes que cada uma dessas regioes possa estar exercendo sobre a regulacao da atividade transcricional do gene da Miostatina, foi realizada uma busca por sitios de ligacao para fatores transcricionais que tenham sido conservados evolutivamente nessas RECs. Muitos sitios conservados foram observados nas sete RECs downstream ao gene da Miostatina, entre eles estao sitios para fatores relacionados ao desenvolvimento de musculatura esqueletica (MyoD, Myogenin, E47, EN1), membros (Pax3, Tbx5) e coracao (Nkx2.5, Pitx2). Juntos, esses dados sugerem uma regulacao modular do gene da Miostatina durante a embriogenese dos vertebrados. A unica REC localizada upstream ao GDF8 representa o promotor minimo putativo deste gene. Essa hipotese e reforcada pela presenca de um sitio de ligacao conservado para a Proteina de Ligacao ao sitio TATA. Com o intuito de validar as hipoteses formuladas com base nas analises de Bioinformatica, no presente trabalho buscamos caracterizar funcionalmente o promotor minimo do gene da Miostatina. Para tanto, a regiao do promotor minimo foi inicialmente clonada em um vetor que nao contem promotor e possui como gene reporter o GFP. Essa construcao de expressao foi entao testada atraves de experimentos de eletroporacao em embrioes de galinha in ovo. A analise dos embrioes eletroporados revelou que a regiao de DNA elegida para as analises funcionais e capaz de dirigir a transcricao do gene reporter, indicando que ela corresponde ao promotor minimo do gene da Miostatina. Alem do sitio TATA, ha, na regiao do promotor, diversos sitios conservados para a ligacao de proteinas envolvidas na via de sinalizacao mediada por cAMP (CREB, ATF, NFY). Esse achado esta de acordo com estudos recentes que demonstram o envolvimento do cAMP na regulacao dos fatores miogenicos Myf5 e MyoD, bem como de Pax3, sugerindo que a atividade do gene da Miostatina tambem possa estar sendo regulada por essa via de sinalizacao. Outras regioes do genoma humano que possuem arquitetura semelhante a observada no promotor da Miostatina foram identificadas, demonstrando que outros genes podem estar sob influencia da mesma via de sinalizacao que regula a atividade do promotor da Miostatina, dentre eles genes envolvidos na miogenese e neurogenese. / Abstract: The Myostatin protein (also known as GDF8) is a member of the transforming growth factor-ß (TGF-ß) superfamily and is expressed almost exclusively in skeletal muscle, both in the embryo and in the adult, where the protein circulates in the blood flow. It was initially identified in 1997 by MCPHERRON et al., and since then many studies have been demonstrating its essential role in the regulation of the development of skeletal muscle from birds and mammals. The knockout of the Myostatin gene causes both hyperplasia and hypertrophy of the skeletal muscle fibers, resulting in muscles twice as big as the wildtype ones, thus showing that Myostatin is a negative regulator of skeletal muscle deposition. The GDF8 structure and function is conserved in many species, including humans where the Myostatin levels are increased during dystrophy conditions and in the cachexia that accompanies some types of cancer and AIDS. A better understanding of the mechanisms that rule the Myostatin expression is essential for the development of strategies that might regulate its activity during such conditions. In this research, we have identified, with the use of bioinformatic tools, the cis-regulatory elements (promoter and enhancers) that regulate the Myostatin gene transcription. We compared the GDF8 loci from human, chicken and mouse and found different evolutionary conserved regions (ECRs), adjacent to the GDF8 coding sequence. Because these intergenic sequences remained relatively conserved throughout evolution, they supposedly have a functional role, possibly as cis-regulatory elements for the Myostatin gene. Our analyses revealed the presence of seven possible enhancers downstream of the GDF8 gene and one conserved region upstream of it. In order to understand the role these regions might have in the regulation of Myostatin's transcription activity, we searched for binding sites that were also evolutionary conserved. Many conserved binding sites were observed in the RECs downstream to the Myostatin gene, and among them are sites for factors related to the development of the skeletal muscle (MyoD, Myogenin, E47, EN1), limbs (Pax3, Tbx5) and heart (Nkx2.5, AREB6, Pitx2). Together, these data suggest a modular regulation of the Myostatin gene during vertebrates' embryogenesis. The only REC observed upstream of the Myostatin locus represents the putative basal gene promoter. This hypothesis is strengthened by the presence of a binding site for the Tata Binding Protein conserved for the studied species. In this research, we aimed at functionally characterizing the Myostatin gene basal promoter. For that purpose, we cloned the studied region in a promoterless vector, which contains GFP as a reporter gene. This expression construct was then tested through in ovo electroporation assays. The analysis of the electroporated embryos revealed that the cloned DNA region is capable of driving the transcription of the reporter gene, which indicates that it truly corresponds to the basal promoter of the Myostatin gene. Moreover, there are conserved binding sites for the CREB and ATF1 transcription factors in the basal promoter, which are activated by the cAMP signaling path. This finding is in agreement with recent studies that demonstrate the involvement of cAMP in the regulation of the myogenic factors Myf5 and MyoD, as well as Pax3, thus suggesting that the activity of the Myostatin gene might be under the influence of this signaling path. Other regions of the human genome that have a similar architecture to the one observed in the Myostatin promoter were identified. This demonstrates that other genes are possibly under the influence of the same signaling path regulating the activity of the Myostatin promoter, among them genes involved in myogenesis and neurogenesis. / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

Orientador: Lucia Elvira Alvares / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T18:28:57Z (GMT). No. of bitstreams: 1 Grade_CarlaVermeulenCarvalho_D.pdf: 3007033 bytes, checksum: 522d9d385305f8744861d38f7dabf5aa (MD5) Previous issue date: 2013 / Resumo: A Miostatina é um regulador negativo da deposição de musculatura esquelética e mutações no gene que codifica esta proteína têm sido associadas a um aumento marcante na massa muscular de organismos vertebrados, resultado de hiperplasia e hipertrofia das fibras musculares. Nosso grupo identificou previamente o promotor basal do gene da Miostatina e análises de bioinformática revelaram a presença de sítios evolutivamente conservados para a ligação de CREB, Meis, FXR e NFY, além de um sítio TATA. No presente trabalho nós utilizamos mutagênese sítio-dirigida para gerar diversas construções delecionais que possuem um ou mais sítios mutados, e testamos sua atividade in vitro usando mioblastos C2C12 de camundongo sob condições de proliferação e diferenciação, para analisar o papel destes sítios de ligação sobre a atividade do promotor. Os resultados mostraram que FXR aparentemente não confere efeito na atividade transcricional do promotor da Miostatina em ambos os momentos analisados, indicando que o papel regulador desta proteína pode estar relacionado ao controle da expressão da Miostatina em outro tipo celular, que não o mioblasto. O NFY apresentou um papel de ativador transcricional, enquanto CREB e Meis atuaram inicialmente como repressores durante a proliferação, passando a relaxar esta repressão durante a diferenciação dos mioblastos, permitindo que a atividade do promotor aumentasse significativamente. Trabalhando juntos, estes fatores de transcrição são capazes de manter a atividade do promotor em níveis mais baixos durante a proliferação dos mioblastos e, com o início da diferenciação, a repressão é liberada, e os níveis de atividade podem aumentar. Este padrão está de acordo com o padrão de expressão dinâmico observado para a proteína da Miostatina durante o desenvolvimento da musculatura esquelética em vertebrados / Abstract: Myostatin is a negative regulator of skeletal muscle deposition and mutations in the gene that encodes this protein have been associated to a remarkable increase in skeletal muscle mass, attributable to both hyperplasia and hypertrophy. We have previously identified Myostatin's basal promoter and bioinformatic analyses revealed the presence of evolutionarily conserved binding sites for CREB, Meis, FXR and NFY, besides a TATA box. In the present study we used site-directed mutagenesis to generate several expression constructs possessing one or more mutated sites, and tested their activity in vitro using mouse C2C12 myoblasts in proliferation and differentiation conditions, to analyze the role of these sites on the activity of the promoter. The results show that FXR appears not to confer any effect on the transcriptional activity of the promoter in both conditions, indicating that the regulatory role of this protein might be involved in the control of Myostatin expression in another cell type. NFY presents a role as transcriptional activator, while CREB and Meis act initially as repressors during proliferation, releasing this repression upon differentiation, which allows the activity of the promoter to significantly increase. Working together, these transcription factors are capable of maintaining the promoter activity at lower levels during the proliferation of myoblasts and, upon differentiation, the repression is released, and activity levels can be increased. This pattern is in agreement with the dynamic expression pattern observed for Myostatin during the skeletal muscle development in vertebrates / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

La myostatine, un facteur de croissance de la famille des TGF-β dont la voie de signalisation agit via l'Activine récepteur de type IIB (ActRIIB), a été identifié comme un régulateur négatif important de la croissance du muscle squelettique. Toutefois, son effet sur le métabolisme énergétique musculaire et sur la fonction du muscle reste largement inexploré. Dans mes travaux de thèse, j'ai étudié la conséquence de l'inhibition de la voie de signalisation ActRIIB sur le métabolisme musculaire, et ceci dans deux modèles expérimentaux, i) les souris constitutives knock-out myostatine et ii) après l'administration pharmacologique de l'ActRIIB soluble chez les souris adultes. Nos résultats démontrent que les souris knock-out myostatine développent une forte fatigabilité, une diminution de la respiration mitochondriale et une signature moléculaire qui tend vers un métabolisme glycolytique. Comme ces résultats peuvent s'expliquer par une conversion congénitale vers des fibres musculaires glycolytiques rapides chez ces souris, j'ai étudié l'effet de l'inhibition de la voie de signalisation ActRIIB chez la souris adulte. J'ai fourni des preuves, notamment pour la souris mdx, modèle animal de la myopathie de Duchenne, que l'inhibition de l'ActRIIB, malgré une distribution de typage de fibres qui reste normale, conduit à une intolérance extrême à l'exercice. Cela a été associé à une augmentation pathologique des taux de lactate sérique ainsi que des caractéristiques prononcées de la myopathie. Plus en détail, l'analyse biochimique et moléculaire montre que l'inhibition de la voie de signalisation ActRIIB diminue l'expression de la protéine porine, réduit la capillarisation musculaire et provoque une déficience de la phosphorylation oxydative. Je montre aussi que l’ActRIIB régule les composants clés du métabolisme musculaire, comme PPARß, Pgc1α, et PDK4, optimisant ainsi les différentes composantes du métabolisme énergétique musculaire. En somme, mes résultats démontrent que l’inhibition de l’ActRIIB provoque une myopathie métabolique, en particulier dans le contexte d’un muscle dystrophique, chez lequel un stress métabolique sous-jacent existe déjà. En conclusion, je ne peux pas recommander l'utilisation de l’inhibition de la voie de signalisation de l’ActRIIB comme stratégie thérapeutique pour les maladies musculaires. / Myostatin, a growth factor of the TGF-β family that signals through the activin receptor-IIB (ActRIIB), has been identified as an important negative regulator of skeletal muscle growth. However, its effect on muscle energy metabolism and energy dependent muscle function remains largely unexplored. I here investigated the consequence of impaired ActRIIB signaling for muscle metabolism in two experimental models, i) the constitutive myostatin knockout mice and ii) following pharmacological administration of soluble ActRIIB in adult mice. Our results demonstrate that myostatin knockout mice develop a strong fatigability, a decrease in mitochondrial respiration and a molecular signature towards a glycolytic metabolism. As these findings may be explained by the congenital shift towards fast glycolytic muscle fibers in these mice, I investigated the effect of inhibition of ActRIIB signaling in adult mice. I provide evidence, notably for the mdx mouse, model for Duchenne muscular dystrophy, that ActRIIB blockade, despite an unchanged fiber type distribution, leads to extreme exercise intolerance. This was associated with pathologically increased serum lactate levels and myopathic features. In-depth biochemical and molecular analysis demonstrates that blockade of ActRIIB signaling down-regulates the ATP channel porin, reduces muscle capillarization and leads to a consecutive deficiency in oxidative phosphorylation. I also show that ActRIIB regulates key determinants of muscle metabolism, such as Pparβ, Pgc1α, and Pdk4, thereby optimizing different components of muscle energy metabolism. Taken together, my results demonstrate that ActRIIB blockade provokes a metabolic myopathy, especially in the context of dystrophic muscle, in which an underlying metabolic stress already exists. In conclusion, I cannot recommend the use of ActRIIB signaling blockade as a therapeutic strategy for muscle diseases.

GASP-1 et GASP-2 sont deux protéines très proches structuralement caractérisées par plusieurs modules inhibiteurs de protéases et identifiés comme des inhibiteurs de plusieurs membres de la famille TGF-ß tel que la myostatine ou GDF-11, respectivement régulateurs négatifs de la myogenèse et de l’ostéogénèse. Malgré l’organisation structurale commune des protéines GASPs, leurs profils d’expression différents laissent supposer des rôles physiologiques distincts. C’est pourquoi, nous avons généré des modèles souris Tg(Gasp-1) et Tg(Gasp-2) surexprimant Gasp-1 ou Gasp-2 afin de mieux appréhender les fonctions de ces protéines. Des analyses fonctionnelles, réalisées in vitro et in vivo dans les contextes musculaire, squelettique et anti-protéasique, nous ont permis de mettre en lumière une dualité fonctionnelle entre GASP-1 et GASP-2 dans ces différents contextes. Les deux lignées Tg(Gasp-1) et Tg(Gasp-2) présentent une augmentation de la masse musculaire due à une hypertrophie sans hyperplasie des myofibres. Cependant, les souris Tg(Gasp-1) présentent une dérégulation globale de l’homéostasie du glucose et des défauts métaboliques en vieillissant, phénotype non retrouvé chez les souris Tg(Gasp-2). Ces résultats nous a permis de proposer la protéine GASP-2 comme étant un meilleur candidat thérapeutique dans le cas de maladies musculaires. Dans le contexte squelettique, seule la surexpression de Gasp-2 entraine un phénotype osseux. L’ensemble de nos résultats permet de mettre en évidence un réseau de régulation de l’expression génique des membres de la famille TGF-ß et de leurs inhibiteurs dans le muscle. / GASP-1 and GASP-2 are two closely related proteins structurally characterized by several protease inhibitor modules and identified as inhibitors of several members of the TGF-ß family such as myostatin or GDF-11, negative regulators of myogenesis and osteogenesis, respectively. Despite the common structural organization of GASPs proteins, their different expression profiles suggest distinct physiological roles. We generated Tg(Gasp-1) and Tg(Gasp-2) mouse models overexpressing Gasp-1 or Gasp-2 in order to better understand the functions of these proteins. Functional analyses, carried out in vitro and in vivo in muscular, skeletal and anti-proteasic context, allowed us to highlight a functional duality between GASP-1 and GASP-2 in the different contexts. Both lines Tg(Gasp-1) and Tg(Gasp-2) exhibit an increase in muscle mass due to myofiber hypertrophy without hyperplasia. However, Tg(Gasp-1) mice have an overall deregulation of glucose homeostasis and metabolic defects with age, a phenotype not found in Tg(Gasp-2) mice. These results allowed us to propose the GASP-2 protein as a better therapeutic candidate for muscle diseases. In the skeletal context, only the Gasp-2 overexpression leads to a bone phenotype. Altogether, our findings highlighted a gene expression regulatory network of TGF-ß members and their inhibitors in muscle.